EP2893019B1 - Sirna and its use in methods and compositions for the treatment and/or prevention of eye conditions - Google Patents

Sirna and its use in methods and compositions for the treatment and/or prevention of eye conditions Download PDF

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EP2893019B1
EP2893019B1 EP13759707.6A EP13759707A EP2893019B1 EP 2893019 B1 EP2893019 B1 EP 2893019B1 EP 13759707 A EP13759707 A EP 13759707A EP 2893019 B1 EP2893019 B1 EP 2893019B1
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sirna
seq
eye
administration
trpv1
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EP2893019A1 (en
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Ana Isabel Jimenez Anton
Victoria GONZALEZ FAJARDO
Veronica RUZ PALOMAR
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Sylentis SA
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/04—Artificial tears; Irrigation solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification

Definitions

  • the present invention relates to the provision of siRNA products and their use in methods and compositions for the treatment and/or prevention of eye conditions related to high levels of expression and or activity of the transient receptor potential vanilloid (TRPV1) using RNA interference.
  • TRPV1 transient receptor potential vanilloid
  • eye conditions associated to ocular pain such as discomfort and altered sensitivity of the cornea following refractive surgery, use of contact lenses, dry eye syndrome, and Sjogren's syndrome, are to be mitigated.
  • RNA interference is a naturally occurring regulatory mechanism of most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans ⁇ Fire, 1998 ⁇ was awarded the Nobel prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells, not through long dsRNAs but by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long ⁇ Elbashir, 2001 ⁇ .
  • siRNAs double-stranded small interfering RNAs
  • RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse phyla and flora, where it is called post-transcriptional gene silencing. Since the discovery of RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise "undruggable" with traditional pharmaceutical approaches involving small molecules or proteins.
  • RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer.
  • the protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) ⁇ Collins, 2005 ⁇ and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3' overhangs and a 5' phosphate and 3' hydroxyl group.
  • PAZ RNA-binding so-called Piwi/Argonaute/Zwille
  • dsRBD double-stranded RNA binding domain
  • RNA-induced silencing complex RNA-induced silencing complex
  • AGO2 endonuclease Argonaute 2 ⁇ Liu, 2004; Song, 2004 ⁇ .
  • AGO2 belongs to the highly conserved Argonaute family of proteins.
  • Argonaute proteins are ⁇ 100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains ⁇ Cerutti, 2000 ⁇ .
  • the PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs ⁇ Song, 2004 ⁇ .
  • AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5' end ⁇ Elbashir, 2001 ⁇ .
  • An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex ⁇ Rand, 2005 ⁇ .
  • siRNA stability enhancement has been the use of modified nucleotides such as 2'-O-methyl nucleotides, 2'-amino nucleotides, nucleotides containing 2'-O or 4'-C methylene bridges.
  • TRPV1 The Transient Receptor Potential Vanilloid-1 (TRPV1), also called Vanilloid Receptor 1 (VR-1), is a capsaicin-responsive ligand-gated cation channel, that was first discovered in 1997 (Caterina, 1997). TRPV1 is mainly expressed on sensory neurons and serves as a molecular detector for heat, capsaicin, protons, and endovanilloids (Caterina, 2001; Montell, 2002; Baumann, 2000). Although the inventors of the present application have also found TRPV1 expression in tissues from the lacrimal gland and ciliary body.
  • TRPV1 When TRPV1 is activated by agonists such as capsaicin and other factors such as heat, acidosis, lipoxygenase products or anandamide, calcium enters the cell and pain signals are initiated. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, and sometimes, in smooth muscle contraction and cough.
  • agonists such as capsaicin and other factors
  • other factors such as heat, acidosis, lipoxygenase products or anandamide
  • calcium When agonists such as capsaicin and other factors such as heat, acidosis, lipoxygenase products or anandamide, calcium enters the cell and pain signals are initiated. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, and sometimes, in smooth muscle contraction and cough.
  • TRPV1 is a known target for treatments by analgesia in response to pain stimul
  • Polymodal nociceptors are the most abundant nociceptor type found in the cornea. There exists pharmacological evidence that these receptor fibers express TRPV1 receptor because they respond to capsaicin, heat and acid. Moreover, high doses of capsaicin inactivate the response of corneal polymodal nociceptors to heat and acid whereas mechanical responsiveness remains unaffected. This suggests that TRPV1 receptors present in corneal polymodal nerve endings were selectively inactivated.
  • TRPV1 activation an important part of the acute nociceptive response to corneal injury and the sustained pain sensations that accompany inflammatory and irritative processes in this tissue are mediated by TRPV1 activation.
  • WO2007/045930 describes the use of TRPV1 specific siRNAs for treatment of ocular pathologies related to ocular pain and dry eye syndrome.
  • the present invention provides improved products for reducing TRPV1 expression and consequent ocular discomfort.
  • the advantage of treating these conditions with siRNA products vs traditional chemical inhibitors is that treatments based on siRNA will have a longer-lasting effect. This result is due to the fact that once the effector molecule is no longer present, the cell will have to synthesise new receptors from scratch; whereas traditional treatments would leave the levels of receptors on the cell membrane intact.
  • WO2011/148193 describes siRNAs which inhibit TRPV1 gene expression, which target a sequence common to transcript variants of TRPV1, 5'-AAGCGCATCTTCTACTTCA-3'.
  • the present invention provides an siRNA targeted to SEQ ID NO: 1 for use in the treatment of dry eye and/or ocular pain wherein the siRNA is administered at a dosage of from about 0.3 to about 0.9 mg per day. Further aspects of the invention are set forth in the dependent claims. Also provided is a medical kit for treatment of dry eye and/or ocular pain, in accordance with independent claim 8.
  • Described herein is the provision of a dosage regimen for an siRNA molecule wherein said molecule specifically targets SEQ ID NO: 1 and reduces expression of the TRPV1 gene when introduced in a cell.
  • a gene is "targeted" by a siRNA according to the present invention when, for example, the siRNA molecule selectively decreases or inhibits the expression of the gene.
  • the phrase "selectively decrease or inhibit” as used herein encompasses siRNAs that affect expression of one gene, in this case TRPV1.
  • a siRNA targets a gene when the siRNA hybridizes under stringent conditions to the gene transcript, i.e. its mRNA. Capable of hybridizing "under stringent conditions” means annealing to the target mRNA region, under standard conditions, e.g., high temperature and/or low salt content which tend to disfavor hybridization.
  • a suitable protocol (involving 0.1 ⁇ SSC, 68 °C for 2 hours) is described in Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, at pages 387-389 .
  • nucleic acid sequences cited herein are written in a 5' to 3' direction unless indicated otherwise.
  • the term “nucleic acid” refers to either DNA or RNA or a modified form thereof comprising the purine or pyrimidine bases present in DNA (adenine "A”, cytosine “C”, guanine “G”, thymine “T") or in RNA (adenine "A”, cytosine “C”, guanine “G”, uracil “U”).
  • Interfering RNAs provided herein may comprise "T" bases, for example at 3' ends, even though "T” bases do not naturally occur in RNA. In some cases these bases may appear as "dT” to differentiate deoxyribonucleotides present in a chain of ribonucleotides.
  • target sequence as defined above is described as a target DNA sequence as used for definition of transcript variants in databases used for the purposes of designing siRNAs, whereas the specific compounds to be used will be RNA sequences defined as such.
  • GenBank Accession Numbers corresponding to four TRPV1 transcripts produced by alternative splicing are: NM_080704 (NM_080704.3, GI:117306161), NM_018727 (NM_018727.5, GI:117306160), NM_080706 (NM_080706.3, GI:117306163) and NM_080705 (NM_080705.3, GI:117306162).
  • ENSEMBL MBL-EBI/Wellcome Trust Sanger Institute
  • TRPV1 transcripts published: ENST00000174621, ENST00000310522, ENST00000344161, ENST00000399752, ENST00000399756, ENST00000399759, ENST00000425167.
  • siRNAs which inhibit TRPV1 gene expression, these siRNAs being especially efficient compared to those already disclosed in the state of the art. Especially efficient meaning that they achieve higher degrees of inhibition and/or a more prolonged effect in time.
  • siRNAs are designed against a target sequence common to all transcript variants of TRPV1 described in the preceding paragraph, and thus mediate RISC-mediated degradation of all possible mRNAs present in the cell encoding TRPV1 protein.
  • Said preferred target region is identified in SEQ ID NO: 1 (5'-AAGCGCATCTTCTACTTCA-3'). They are described in WO2011/148193 .
  • a siRNA according to the aspects of the present invention comprises a double stranded RNA molecule, whose antisense strand will comprise or consist of an RNA sequence substantially complementary to SEQ ID NO: 1, and its sense strand will comprise an RNA sequence complementary to the antisense strand, wherein both strands are hybridised by standard base pairing between nucleotides.
  • substantially complementary to a target mRNA sequence, may also be understood as “substantially identical” to said target sequence.
  • Identity as is known by one of ordinary skill in the art, is the degree of sequence relatedness between nucleotide sequences as determined by matching the order and identity of nucleotides between sequences.
  • the antisense strand of an siRNA having 80%, and between 80% up to 100% complementarity, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity, to the target mRNA sequence are considered substantially complementary and may be used in the present invention.
  • the percentage of complementarity describes the percentage of contiguous nucleotides in a first nucleic acid molecule that can base pair in the Watson-Crick sense with a set of contiguous nucleotides in a second nucleic acid molecule.
  • RNA interference As is known from the state of the art, many different structures have been proposed to achieve RNA interference. Generally these double stranded molecules are from about 19 to about 25 nucleotides in length, and include blunt-ended structures as well as those with overhangs. Overhangs have been described to be advantageous and may be present on the 5' ends or on the 3' ends of either strand as they reduce recognition by RNAses and imitate Dicer's natural substrate. Some authors recommend including overhangs on both 3' ends of the molecules, whereas others consider one overhang to be sufficient. Others have described the use of blunt-ended structures with specific modification patterns ( EP 1527176 , WO 2008/104978 , and many others).
  • Overhangs may be comprised of between 1 and 5 nucleotides, typically overhangs are made up of dinucleotides.
  • Classical molecules used in the field comprise a 19 nucleotide double stranded molecule which further comprises 3' dinucleotide overhangs preferably comprising deoxynucleotides as taught in initial studies by Tuschl ( WO02/44321 ). These overhangs are said to further enhance resistance to nuclease (RNase) degradation.
  • Kim et al 2005 describe that 21-mer products (containing dinucleotide overhangs) are necessary for loading onto RISC.
  • Bramsen et al. 2009 describe the introduction of possible destabilizing modifications to the overhangs to further increase silencing efficiency.
  • a preferred embodiment of the various aspects of the present invention refers to siRNA molecules targeting SEQ ID NO: 1 which comprise at least one overhang.
  • Another alternative embodiment of the various aspects of the present invention provides blunt-ended molecules.
  • the present invention discloses an siRNA comprising or consisting of a 19 nucleotide double-stranded structure targeting SEQ ID NO: 1. Surprisingly, said 19 nucleotide double-stranded RNAs have proven to be more resistant to degradation than previously described products with 21 nucleotides and 3' overhangs as may be seen in figure 5 .
  • the present invention discloses a 19 nucleotide double-stranded blunt-ended siRNA targeted against SEQ ID NO: 1.
  • this compound is identified as SEQ ID NO: 2 (5'-AAGCGCAUCUUCUACUUCA-3').
  • the antisense strand of this siRNA is at least 80%, preferably at least 90%, complementary to SEQ ID NO: 1.
  • siRNA molecules are their instability in biological fluids due to the ubiquitous nature of RNAses. Consequently, the use of many different chemical modifications to nucleotides has been described with the purpose of enhancing compound stability.
  • siRNAs have been found to induce unspecific activation of the innate immune system, including up-regulation of certain cytokines, e.g. type I and/or type II interferon as well as IL-12, IL-6 and/or TNF-alpha production.
  • cytokines e.g. type I and/or type II interferon as well as IL-12, IL-6 and/or TNF-alpha production.
  • the origin of these effects is thought to be activation of Toll-like receptors such as TLR7, TLR8 and/or TLR3 by siRNA.
  • the siRNA can further comprise at least one nucleotide with a chemical modification.
  • Preferred chemical modifications which enhance stability and reduce immunogenic effects include 2'-O-methyl nucleotides, 2'-fluoro nucleotides 2'-amino nucleotides, 2'-deoxy nucleotides, nucleotides containing 2'-O or 4'-C methylene bridges.
  • a further preferred chemical modification within the meaning of the present invention relates to the substitution of uracyl ribonucleotides with deoxythymidine (deoxyribonucleotides).
  • the at least one chemically modified nucleotide is on the sense strand, on the antisense strand or on both strands of the siRNA.
  • the siRNA can be selected from SEQ ID. NO.3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • siRNA molecules as described above may be delivered to the cell interior in their native structure using methods known in the art. For example, when studying in vitro gene silencing, these compounds are administered using standard transfection reagents. To achieve effects in vivo these compounds may also be administered naked or using delivery enhancing agents such as for example liposomes, conjugation with a specific moiety, etc. although many different alternatives are known in the art, and are used differently depending on the desired target site within the body.
  • siRNA molecules of the various aspects of the invention can be expressed within cells from eukaryotic promoters.
  • Recombinant vectors capable of expressing the siRNA molecules can be delivered and persist in target cells.
  • vectors can be used that provide for transient expression of nucleic acid molecules. Such vectors can be repeatedly administered as necessary.
  • the siRNA molecule interacts with the target mRNA and generates an RNA interfering response.
  • shRNA short hairpin RNA
  • siRNA molecule expressing vectors can be systemic, such as by intravenous or intra-muscular administration, by administration to target cells ex-planted from a subject followed by reintroduction into the subject, or by any other means that would allow for introduction into the desired target cell.
  • siRNA targeting SEQ ID NO.1 in the preparation of a medicament for use in a method of treatment of an eye condition characterised by increased expression and/or activity of TRPV1 wherein the siRNA is administered according to the dosage regimen disclosed herein.
  • the method comprises inhibiting expression of TRPV1 in a patient.
  • inhibition is used to indicate a decrease or downregulation of expression or activity.
  • the eye condition is ocular pain.
  • the eye condition is selected from the group comprising ocular discomfort and altered sensitivity of the cornea following refractive surgery, use of contact lenses, dry eye syndrome, Sjogren's syndrome, and other eye pathologies.
  • Therapeutic treatment with siRNAs directed against TRPV1 mRNA is expected to be beneficial over small molecule topical ocular drops by increasing the length of time that effect is observed, thereby allowing less frequent dosing and greater patient compliance. This is especially important in cases such as dry eye syndrome and altered corneal sensitivity as they are often chronic conditions.
  • the siRNA described herein may be formulated.
  • the compositions and formulations of said siRNAs may be administered topically to the organ of interest.
  • they may be formulated for topical administration to the eye, preferably to the corneal surface of the eye.
  • Application to the corneal surface may, for example be in the form of eyedrops, a gel, lotion, cream or ocular inserts.
  • Other administration forms to the eye may include injection into the eye.
  • siRNA specifically targeting SEQ ID NO: 1 as described in the preceding paragraphs, for use as a medicament for the treatment of an eye condition characterised by increased expression and/or activity of TRPV1 wherein the siRNA is administered according to the dosage regimen disclosed herein.
  • it may be an siRNA comprising or consisting of a 19 nucleotide double-stranded structure targeting SEQ ID NO: 1. This siRNA may be blunt-ended.
  • the siRNA is SEQ ID NO: 2.
  • Other siRNA for use may be selected from SEQ ID. NO.3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
  • the siRNA of the invention preferably comprises at least the same seed sequence.
  • any sequence that specifically targets SEQ ID No.1 is preferably identical in positions 2-8 of the antisense strand.
  • siRNAs described herein may be used to silence TRPV1 expression in tissues other than the eye. Consequently, said siRNAs should be formulated accordingly.
  • a siRNA molecule can comprise a delivery vehicle, including liposomes, for administration to a subject.
  • Carriers and diluents and their salts can be present in pharmaceutically acceptable formulations.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as biodegradable polymers, hydrogels, cyclodextrins poly (lactic-co-glycolic) acid (PLGA) and PLCA microspheres, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors.
  • PLGA cyclodextrins poly (lactic-co-glycolic) acid
  • the nucleic acid molecules described herein can also be formulated or complexed with polyethyleneimine and derivatives thereof, such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • polyethyleneimine and derivatives thereof such as polyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL) or polyethyleneimine-polyethyleneglycol-tri-N-acetylgalactosamine (PEI-PEG-triGAL) derivatives.
  • PBS phosphate-buffered saline
  • a siRNA molecule described herein may be complexed with membrane disruptive agents and/or a cationic lipid or helper lipid molecule.
  • Delivery systems which may be used include, for example, aqueous and non-aqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and non-aqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e. g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e. g. , polycarbophil and polyvinylpyrolidone).
  • the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
  • a pharmaceutical formulation described herein is in a form suitable for administration, e.g., systemic or local administration, into a cell or subject, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • compositions prepared for storage or administration that include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art.
  • preservatives, stabilizers, dyes and flavouring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state.
  • the pharmaceutically effective dose generally depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize.
  • the administration schedule is an important determinant of effective downregulation by siRNA molecules.
  • the inventors have developed an effective dosage schedule for administration of a siRNA for the treatment of an eye condition characterised by increased expression and/or activity of TRPV1, in particular dry eye and or ocular pain, which avoids side effects and can be safely administered.
  • administration of an siRNA molecule wherein said molecule specifically targets SEQ ID NO: 1 and reduces expression of TRPV1 gene when introduced in a cell according to the dosage regimens described herein leads to clinical improvement.
  • an "effective dosage schedule” refers to the amount of siRNA sufficient to treat or manage an eye disorder associated to overexpression of TRPV1.
  • an "effective dosage schedule” refers to the amount of siRNA sufficient to treat or manage an eye disorder associated to overexpression of TRPV1.
  • OSDI ocular surface index
  • VAS visual analogical scale
  • Any reduction in these levels as compared to pretreatment levels is advantageous, whether the compounds are delivered alone, or in combination with another suitable therapeutic. (e.g., the disclosure contemplates a decrease in OSDI and/or VAS greater than about 5%, about 10%, about 25%, about 30%, about 35%, about 40%, about 50%, or about 60% of pretreatment IOP).
  • a therapeutically effective amount may also refer to the amount of an siNA sufficient to delay or minimize the onset of an eye disorder associated with dry eye and/or ocular pain.
  • a therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of an eye disorder associated with dry eye and/or ocular pain.
  • a therapeutically effective amount with respect to an siNA of the invention means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of an eye disorder associated with dry eye and/or ocular pain. Used in connection with an amount of an siNA, the term can encompass an amount that improves overall therapy, reduces or avoids unwanted effects, or enhances the therapeutic efficacy of or synergizes with another therapeutic agent.
  • a therapeutic benefit in the treatment or management of an eye disorder such as dry eye and/or ocular pain is the sustained decrease in pain and/or undesirable sensations.
  • siRNA will decrease the levels of TRPV1 receptors within the cell, once the treatment stops the cell must re-synthesise new receptors before pain sensations will be perceived.
  • therapies based on siRNA treatments will have a more sustained effect. This is considered a significant enhancement of the therapeutic efficacy.
  • siRNA is the minimum probability of side effects or acute toxicity issues derived from its presence in systemic circulation, often associated with different eyedrop-based treatments. This is due to the fact that when the compound enters the bloodstream, it will be rapidly degraded by RNAses present in the blood.
  • the fact that the formulation described herein can be offered in single dose vials means incorporation antimicrobial preservatives, present in the majority of formulations on the market today, and which produce a certain intolerance in some patients, making it necessary to stop the treatment. Both issues are especially important when bearing in mind that conditions like dry eye and or ocular pain are often chronic and therefore so is the treatment.
  • One of the preferred administration routes is topical, by instillation directly to the eye, preferably using eye drops.
  • therapeutic treatment with siRNAs directed against TRPV1 mRNA is expected to be beneficial over small molecule topical ocular drops by increasing the length of time that effect is observed, thereby allowing less frequent dosing and greater patient compliance.
  • the siRNA is administered directly to the eye, generally an amount of about 0.01 mg to about 100 mg per day and per eye can be administered. In one embodiment, the amount administered per day and per eye is about 0.1 mg to about 10 mg.
  • about 0.04 mg to 80 mg, about 0.04 mg to about 20 mg, about 0.08 mg to about 10 mg, about 0.08 mg to about 1.2 mg, about 0.3 to about 0.9 mg, or about 0.08 mg to about 0.9 mg, per eye per day of siNA is administered.
  • the dosage is about 0.5mg to about 1.5mg. In one embodiment, the dosage is about 0.3 to 0.9 mg, preferably about 0.6 mg to about 0.9 mg. Alternatively a preferred dosage is about 0.6 mg or about 0.9 mg per eye per day.
  • eyedrops One of the preferred administration routes as mentioned above is via the use of eyedrops.
  • these eyedrops have a volume of between 25 and 50 microliters containing the given dose of compound, preferably between 26 and 40 microliters.
  • commercial eyedroppers may be used in the final presentation of the medicine, and the resulting volume would be between about 30 and about 33 microliters per drop.
  • the eyedrops are delivered in a volume of about 40 ⁇ l.
  • the composition of the invention comprises an siRNA such as that of SEQ ID NO: 2 in an acceptable solution such as phosphate-buffered saline at a concentration of from about 7.5 to about 22.5 mg/ml, or alternatively from about 15 mg/ml to about 22.5 mg/ml.
  • the compositions of the invention can comprise the above concentrations of siRNA in PBS and optionally pharmaceutically acceptable excipients such as for example benzalkonium chloride.
  • Treatment at the dosages above may be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days.
  • administration is for 10-15 days, most preferably for 10 days.
  • Administration may be followed by a rest period, for example a rest period of 7 days before continuation of the treatment.
  • the dosages may be administered on a daily basis during a long period resulting in a chronic administration. Accordingly, administration may be continued for more than 4 weeks on a daily basis, or alternatively, administration may be continued for more than 4 weeks but not on a daily basis.
  • the precise schedule can be determined in accordance with the severity of the chronic condition.
  • administration routes other than directly to the eye can also be used.
  • the precise dosage and administration schedule to be employed in the formulation will also depend on the route of administration, but the dosages above can be employed and generally an amount of about 0.01 mg to about 100 mg per day and per eye can be administered.
  • the precise dosage and administration schedule to be employed also depends on the seriousness of the disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. It is also understood that the specific dose level for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavouring agents, colouring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients can be, for example, inert diluents; such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in a mixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • colouring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavouring agents such as sucrose or saccharin.
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents and flavouring agents can be added to provide palatable oral preparations. These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • a dispersing or wetting agent exemplified by those already mentioned above.
  • Additional excipients for example sweetening, flavouring and colouring agents, can also be present.
  • compositions can also be in the form of oil-in-water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavouring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavouring and colouring agents.
  • sweetening agents for example glycerol, propylene glycol, sorbitol, glucose or sucrose.
  • Such formulations can also contain a demulcent, a preservative and flavouring and colouring agents.
  • the pharmaceutical compositions or siRNA described herein can be in the form of a sterile injectable aqueous or oleaginous suspension.
  • This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • a sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • a non-toxic parentally acceptable diluent or solvent for example as a solution in 1,3-butanediol.
  • acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • compositions described herein are formulated in a solution, preferably a buffered saline solution such as PBS, or a gel for topical administration to the eye, such as, for example, in the form of eyedrops.
  • the formulations may be cationic emulsions and/or contain biopolymers including, but not limited to, poly(lactide-co-glycolide), carbopol, hialuronic acid and polyacrylic acid.
  • nucleic acid molecules described herein can also be administered in the form of suppositories, e. g., for rectal administration of the drug.
  • suppositories e. g., for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules described herein can be administered parenterally in a sterile medium.
  • the drug depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anaesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • composition wherein said composition comprises at least an siRNA targeting SEQ ID NO: 1 at a specific dosage schedule, as has been described in the preceding paragraphs.
  • nucleic acid molecules described herein can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect.
  • the use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • kits that comprise a dispenser with an orifice for dispensing specific dosages of the siNA compound in a droplet of predetermined volume.
  • the siNA compounds are siRNAs targeted against SEQ ID NO: 1.
  • the dispensers within the kit provide a composition comprising or consisting of SEQ ID NO: 2.
  • the kit can comprise a collection of single use dispenser, for example for use during one month, in this specific case, the case would contain 30 single use dispensers.
  • the droplet can range from about 50 ⁇ l to about 100 ⁇ l in volume.
  • the dispenser can be a single use dispenser and comprise between about 1 mg and about 2 mg of the siNA compounds, and optionally also comprise one or more pharmaceutically acceptable diluents, and optionally one or more excipients.
  • the composition contained in the dispenser can comprise a concentration of between about 7.5 mg/ml to about 22.5 mg/ml of the siNA compound.
  • the dispenser can be designed to be used for one month or more and the volumes contained will increase accordingly to provide the equivalent number of doses.
  • the kits can also comprise instructions specifying that a dosage of the siRNA compound of between about 0.3 mg and about 0.9 mg in 1 droplet is to be applied to each eye.
  • the instructions can further specify that the droplets are applied to each eye once a day, twice a day, three times a day, or four times a day, and that the application to each eye is to take place daily, every other day, once a week, twice a week, three times a week, every other week, or once a month.
  • siRNAs In order to find a particularly effective target sequence for siRNAs to silence TRPV1 (which obtain important inhibition of gene expression), six different siRNAs were tested. These siRNAs are described as SEQ ID NO: 2, SEQ ID NO: 7 and SEQ ID NO: 17 to 20.
  • HeLa human cervix adenocarcinoma
  • HeLa cells were transfected with 100nM of different compounds and Lipofectamine 2000 as a transfectant agent. All transfections were done following standard manufacturer's conditions. In the same transfection a different scramble siRNA was used as control. Cell pellets were collected at 24, 48, and 72 hours to evaluate possible variations in protein levels and processed by real-time PCR. In order to quantify the results obtained by real-time Qrt-PCR, we used the Comparative Threshold Method.
  • an siRNA directed against target sequence SEQ ID NO: 1 is much more efficient in terms of TRPV1 gene silencing than previously described siRNA products directed against a different region of the same gene. Moreover this effect is sustained in time, as at 72 hours post-transfection there is still significant downregulation of mRNA levels. This duration of the effect is unpredictable and is sequence specific.
  • underline represents bases comprising a 2'-Omethyl group.
  • a further unexpected beneficial effect derived from the above described compounds is their enhanced resistance to degradation by RNases as may be seen in Figure 5 .
  • siRNAs of the present invention are highly conserved region of the TRPV1 gene, throughout different animal sequences. In fact, this sequence is identical between human and rabbit, making this animal model especially suitable for the study of said diseases.
  • Test and reference items were instilled once a day from Day 1 to Day 3 and twice a day on Day 4 (pace out of 60 min) in the right eyes. At Day 4, 15 minutes following the last instillation, corneal pain was induced in the right eye of the animals by a single instillation of capsaicin 1%. The contralateral eye was instilled with PBS throughout the study and served as control.
  • palpebral opening was measured. It is considered that the eye is closed in response to pain, and as pain sensations subside the palpebral opening will increase back to normal levels.
  • the palpebral opening was measured before treatment (baseline), just before pain induction and then 1, 5, 10, 15, 20, 25, 30, minutes after pain induction.
  • a compound was tested, specifically the compound of SEQ ID NO: 2, and was observed to induce a higher analgesic effect than capsazepine (eye recovery as measured by degree of palpebral opening). Therefore this compound has proven to be an effective therapeutic treatment for ocular discomfort.
  • the compounds are much more effective when silencing TRPV1 gene expression in ocular tissues than previously described compounds.
  • the higher efficacy together with the longer lasting effect of the compounds, should provide advantageous dose regimes, as allowing more time between doses would significantly improve patients' quality of life.
  • the ocular tolerance of the compound according to SEQ ID NO: 2 was assessed in 30 healthy human adults.
  • the study was organised into two periods. During the first period an initial safety evaluation was made using a single dose of the investigational product, followed by a second period with a multi-dose administration.
  • Period 1 single dose: controlled with no intervention, and randomisation of the eye receiving the administration.
  • Each volunteer was his/her own control, given the fact that the test item was administered to just one eye whilst the other eye received no intervention, however the safety evaluation tests were conducted.
  • the ophthalmologist making the safety evaluation was blind to the drug administration. Product absorption into the bloodstream was determined.
  • Period 2 multi-dose: Open, parallel and controlled. The treated eye was randomised. The evaluator was blind as to the investigational product administration site.
  • the eye before drug administration and the other eye were both considered as controls.
  • the second stage commenced once the safety and pharmacokinetics of the first stage had been evaluated.
  • the results for the first stage established the need to continue with the pharmacokinetic evaluation during the second stage.
  • Period 1 had 6 volunteers and period 2 had 24 volunteers, divided into two cohorts with a different dose level applied to each half of the group. Period 1 comprises a single dose and period 2 comprises 7 doses in total (one administration per day).
  • Local tolerance assessment was based on the frequency of alterations (local adverse effects) detected on the eye surface during explorations performed 24 hours after last dose administration in Period 2 and 72 hours after the single dose instillation in period 1. Good tolerance was defined as the absence of grade 3 toxicity or higher, on the CTCAEv3 scale (Common Terminology Criteria for Adverse Events).
  • a further object of this trial was to assess systemic tolerance to the compound, by monitoring repercussions on the analytical parameters, on the physical examination, vital signs and the electrocardiogram following treatment.
  • Haemoglobin (g/dl) 13.7 ⁇ 1.4 13.8 ⁇ 1.6 n.s.s. Hematocrit (%) 43.0 ⁇ 4.2 43.0 ⁇ 5.2 n.s.s.
  • MCV (fl) 90.6 ⁇ 4.0 89.8 ⁇ 4.2 0.001* MCH (pg) 28.9 ⁇ 1.6 28.9 ⁇ 1.7 n.s.s.* MCHC (g/dl) 31.9 ⁇ 1.4 32.2 ⁇ 1.1 n.s.s.* Platelets (10 9 /l) 238.9 ⁇ 48.1 240.5 ⁇ 44.3 n.s.s.
  • Urinalysis Density 1.021 ⁇ 0.008 1.023 ⁇ 0.008 n.s.s.* pH 6.3 ⁇ 0.7 6.3 ⁇ 0.6 n.s.s.* n.s.s. : Not statistically significant. * Wilcoxon Test.
  • the vital signs (blood pressure and heart rate) were taken during the selection, and at different times during the treatment phase and in the final examination (see Tables 7 and 8).
  • Blood analysis was performed to determine SEQ ID NO: 2 concentrations in plasma samples obtained following the administration of the drug.
  • sampling was performed over the 4 hours following administration, with blood being extracted at 5, 15, 30 minutes and 4 hours after product administration.
  • blood samples were collected 5 minutes after administration on day 1, and again on day 7 both before compound administration and also 5 minutes after product administration.
  • a pharmacokinetic profile could not be determined for either period, as the compound was not detected in any of the collected blood samples with the validated bioanalytical method (LLOQ: 10 ng/mL).
  • LLOQ validated bioanalytical method
  • a parallel, placebo controlled, double-masked clinical study was designed to evaluate the analgesic effect and tolerability of the compound of SEQ ID NO: 2 administered daily as eye drops during 10 days of treatment. Secondary objectives being assessment of local tolerability after each dose, systemic tolerability (effect on laboratory parameters, physical examination, and vital signs), and changes (if any) in visual acuity, intraocular pressure, Schirmer's test and tear break-up time, possibly related to the investigational product.
  • the drug or placebo is instilled in both eyes. Both eyes are monitored in a blinded fashion.
  • subjects are randomised to compound or placebo in a ratio of 2:1 administered in topically to the eye, as eye drops.
  • Subjects receive a final volume of 40 ⁇ l of compound or vehicle (placebo) per eye.
  • the dose administered is 900 ⁇ g of compound per eye.
  • Subjects return each day (including bank holidays and weekends) to the site for investigational product administration and assessments. Subjects receive 1 dose of the compound once a day in both eyes for 10 days.
  • the final assessment is done at the follow-up visit which takes place 14 to 20 days after the first administration (from 4 to 10 days after the last administration) to determine patients' evolution after finalisation of the treatment period.
  • each individual score on OSDI ⁇ and VAS is taken down the day before beginning treatment, and compared to that of day 10. The result is measured specifically in changes in median score resulting from OSDI ⁇ questionnaire, and changes in median intensity of VAS evaluation.
  • results from ocular explorations performed before initiating treatment and after 10 days are compared to confirm tolerability.
  • the OSDI ⁇ questionnaire is assessed on a scale of 0 to 100, with higher scores representing greater disability.
  • the index demonstrates sensitivity and specificity in distinguishing between normal subjects and patients with dry eye disease.
  • the OSDl ⁇ consists of twelve questions and is designed to provide a quick indication of the symptoms that are consistent with dry eye disease.
  • the OSDI ⁇ is a valid and reliable instrument for measuring the severity of dry eye disease (normal, mild to moderate, and severe) and has been accepted by the Food and Drug Administration for use in clinical trials. The validity and reliability of the OSDI ⁇ have been assessed and it has been found to provide good to excellent reliability, validity, sensitivity and specifity for dry eyes.
  • OSDI ⁇ score defined the ocular surface as normal (0-12 points) or as having mild (13-22 points), moderate (23-32 points), or severe (33-100 points) disease.
  • VAS pain questionary VAS is a unidimensional measure of pain intensity, which has been widely used in diverse adult populations.
  • the VAS measures pain that ranges across a continuum of values and cannot easily be directly measured, like the ocular pain.
  • the scale is most commonly anchored by "no pain” (score of 0) and "worst imaginable pain” (score of 10).
  • V0 corresponds to the VAS the day before beginning the treatment
  • VD10 corresponds to the VAS the day 10.
  • the % shows the changes in median score on patients' level of ocular pain on the right and left eye.

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JP2018138596A (ja) 2018-09-06
KR102136539B1 (ko) 2020-07-23
KR20150047513A (ko) 2015-05-04
EP2893019A1 (en) 2015-07-15
CA2883040C (en) 2023-01-17
WO2014037377A1 (en) 2014-03-13
US9808479B2 (en) 2017-11-07
AU2013311781A1 (en) 2015-03-12
CY1121000T1 (el) 2019-12-11
PT2893019T (pt) 2018-10-19
PL2893019T3 (pl) 2018-12-31
BR112015004469B8 (pt) 2022-12-06
PE20150619A1 (es) 2015-05-11
CL2015000537A1 (es) 2015-07-10
JP6742362B2 (ja) 2020-08-19
IL237389A0 (en) 2015-04-30
ZA201501494B (en) 2016-01-27
GB201215857D0 (en) 2012-10-24
ES2685346T3 (es) 2018-10-08
BR112015004469B1 (pt) 2022-11-16
HUE040096T2 (hu) 2019-02-28
RU2015112121A (ru) 2016-10-27
BR112015004469A2 (pt) 2017-08-08
LT2893019T (lt) 2018-08-10
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IN2015DN02501A (hr) 2015-09-11
RU2663100C2 (ru) 2018-08-01
US20150224131A1 (en) 2015-08-13
DK2893019T3 (en) 2018-09-03
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